West Nile Virus: From Mechanisms to Novel Research Techniques

The West Nile Virus was first introduced to the Unites States in the early 21st century. Along with the rise of this new virus, panic spread as well. However, with the development of many new serologic techniques and the even more recent real-time reverse transcription PCR technique, early and specific detection of this virus is now possible and will lead to fewer epidemics in the future.

Introduction

Outbreaks of the West Nile Virus (WNV) have been reemerging ever since its first documentation in 1937 from the West Nile District of Northern Uganda. From 1999 to 2003, the epidemic spread west across the North American continent. Following a slight drop in 2003, cases reappeared in 2005-2007 and 2012, with the most recent reports occurring in the summer of 20141. Because the West Nile Virus has become well established in North America, infesting a variety of hosts such as people, birds, and horses, it has become a health priority to develop rapid and accurate diagnostic techniques2. It is predicted that since this virus has spread so quickly in the past, it will likely to continue this pattern of dissemination in the future. Early detection can help environmental engineers and other scientists quarantine certain areas to prevent future epidemics3.

Molecular Mechanisms and Pathogenesis

The WNV belongs to the Flavivirus genus and Flaviridae family. Other common pathogenic flaviviruses include Japanese encephalitis (JE), yellow fever (YF), and Murray Valley encephalitis (MVE)4. The spherical viral structure consists of a nucleocapsid surrounded by a lipid bilayer. The single-stranded RNA resides inside the nucleocapsid and is characterized by a positive polarity and an open reading frame ((Shi, Pei Yong, and Susan J. Wong. "Serologic Diagnosis of West Nile Virus Infection." Expert Review of Molecular Diagnostics, November 2003, 733-41. June 26, 2016.)). Flaviviruses need two factors in order to be maintained in nature: a vector and a host. Most commonly, mosquitoes act as the vector and wild avian species act as the host. Through a mosquito-avian transmission cycle, WNV is maintained and amplified5.

Clinical Presentations

Symptoms of WNV vary across different species ranging from severe symptoms to asymptomatic. In birds, infection causes extreme viraemia and inflammation. Shortly afterwards, tissues, internal organs, and the central nervous system (CNS) suffer necrosis and hemorrhaging. Viraemia is lower in horses; however, clinical signs include CNS lesions, muscular fasciculation, and weakness. Humans typically show no symptoms; however, some will display West Nile Fever4. There are currently no treatments available for humans. Severe cases would require hospitalization in order to receive supportive treatment. Although these treatments can reduce pain and discomfort, they do not directly combat the virus.

MAC ELISA

In laboratory studies, MAC ELISAs (IgM antibody capture enzyme-linked immunosorbent assays) have started to become more widely used because of its many advantageous. The technique is sensitive for early detection and avoids the influence of IgG antibodies. After the onset of WNV infection, IgM antibodies are released into the bloodstream. MAC ELISAs are used as an indicator of recent infection and function to detect IgM levels in the bloodstream. In the first step of a MAC ELISA, goat anti-IgM is used as capture antibodies. Blocking of the test plates with nonfat dry milk is important because it reduces nonspecific binding and background. Subsequently, human sera are added to the plate to react with the antihuman IgM, viral antigens are added to react with the serum antibodies, and labeled monoclonal antibody (mAb) is added to react with the antigens. Finally, the substrate tetra methyl benzidine is added. Using a microplate reader, the absorbance of the test serum is compared to the absorbance of the negative control sample. The respective ratio must be greater than 2.0 in order to be considered a positive result. However, many disadvantageous also exist including the need for follow up testing and the possibility of cross-reactions with other similar flaviviruses such as JE or YF6.

Fluorescent Microsphere Immunoassay

A relatively new technique, immunoassays are able to portray zones on nitrocellulose membranes by using microspheres with bound antigens and lateral flow immunochromatography technology. After incubation of human or horse samples, the antibodies that are bound to the microspheres can be detected with species-specific secondary antibodies. In comparison with ELISAs, microsphere immunoassays have several advantages. This technique is more quantitative; requires shorter time due to the shaking motion during incubation; and the analysis has higher precision, which calls for less valuable specimen volumes7. Purified WNV recombinant E, NS3, and NS5 proteins were tested with this method. The NS3 protein was found to produce unreliable results from the test; however, the E and NS5 proteins demonstrated the capacity to be used as reliable diagnostic tools for humans in the future8.

RT-qPCR

Reverse transcription quantitative polymerase chain reaction is the most recent diagnostic technique. This technique transcribes the virus RNA into DNA with the enzyme reverse transcriptase. After producing the DNA template and the annealing of two primers, extension occurs. Through many subsequent cycles, the DNA is amplified to the desire amount9. The primer is sequence specific to the WNV RNA, so if amplification occurs the test is positive, but if amplification fails, the test is negative10.However, one disadvantage is that negative results do not guarantee that the serum is not infected with WNV11.

Future Outlook

MAC ELISAs and microsphere immunoassays will continue to play an important role for human diagnostic tests. One advantage of the microsphere immunoassay is its ability to detect multiple antigens in a single test. A disadvantage is the inability to differentiate between the similar flaviviruses that have infected the same geographical location due to cross-reaction. One solution was the development of RT-qPCR, a nucleic-acid based assay that targets the RNA sequences of the WNV virus (("West Nile Virus: Detection with Serologic and Real-time PCR Assays." Quest Diagnostics. Quest Diagnostics, 2013. Web. 24 June 2016., "WNV Antibody Testing." Centers for Disease Control and Prevention. February 12, 2015. Accessed July 24, 2016.)).However, negative results require follow up testing for confirmation. Therefore, new techniques should be developed that do not cross react to use along with RT-qPCR. Furthermore, currently there are no vaccine or cures against this virus for humans. The only successes in research have been in vaccine development for horses and only supportive care exists for humans. Therefore, a pressing current priority is to develop a WNV vaccine that is specific to humans.